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Proteolytic degradation of I-kappa-B-alpha is essential for activation of NF-kappa-B (1-4). When degradation is blocked by certain protease inhibitors that target proteasomes, activation of NF-kappa-B is prevented. Physiologic stimulation also induces phosphorylation of I-kappa-B-alpha (1-7), but the significance of this phosphorylation for activation of NF-kappa-B in cells has remained unclear. Induced phosphorylation does not in itself dissociate complexes of I-kappa-B-alpha and NF-kappa-B in vivo (1, 2, 4, 7). The question arises as to how proteolysis of I-kappa-B-alpha is triggered.
To identify regions in I-kappa-B-alpha essential for signaling and degradation, we systematically mutated the human I-kappa-B-alpha gene and stably transfected the altered genes into mouse EL-4 T lymphocytes (8-10). Human I-kappa-B-alpha was distinguished from endogenous murine I-kappa-B-alpha by its slower mobility on SDS gels. The exogenous human wild-type I-kappa-B-alpha and endogenous murine I-kappa-B-alpha were degraded with similar kinetics upon cellular stimulation with tumor necrosis factor-alpha (TNF-alpha) (Fig. 1A) or phorbol 12-myristate 13-acetate (PMA) + ionomycin (Fig. 1B). (Figures 1A and 1B omitted) Furthermore, the human I-kappa-B-alpha was phosphorylated in a signal-dependent manner, as indicated by the characteristic shift in electrophoretic mobility that accompanies signal-induced phosphorylation of endogenous I-kappa-B-alpha from human cells (2) (Fig. 2, A and D). (Figure 2, A and D omitted) To block proteolysis in the latter experiments, we added calpain inhibitor I to the cells (11). Although murine I-kappa-B-alpha was also phosphorylated upon stimulation (see below), only a small change in mobility was observed. The proteolysis and phosphorylation tests established that permanently transfected human wild-type I-kappa-B-alpha is fully signal-responsive in these cells.
We then tested various deletions and point mutants of human I-kappa-B-alpha in EL-4 cells (10). A deletion mutant missing the NH sub 2 -terminal 54 amino acids (Delta-N) was neither proteolyzed nor phosphorylated in response to signals (Figs. 1B and 2B), but still fully inhibited NF-kappa-B (Fig. 3A). (Figures 2B and 3A omitted) We therefore investigated potential phosphoacceptor sites within the deleted portion. I-kappa-B-alpha containing a Ser sup 32 -->Gly mutation (mt sup 32 ) was not degraded upon stimulation by TNF-alpha or PMA + ionomycin (Fig. 1D) (12) nor did it undergo the characteristic mobility shift upon cellular stimulation, suggesting a defect in signal-induced phosphorylation (Fig. 2D). (Figure 2D omitted) I-kappa-B-alpha containing a Ser sup 36 -->Ala mutation (mt sup...